Blood glucose concentration is a very important indicator of diabetes. Determination of blood glucose concentration is extremely important for clinical diagnosis and management of diabetes. Detection of blood glucose generally involves glucose oxidation catalyzed by an enzyme. The enzyme can be divided into glucose oxidase (GOD) and glucose dehydrogenase (GDH) according to enzyme types. When blood glucose is detected by means of a test strip and a glucometer, although GOD has a relatively higher specificity and is not interfered by sugars other than glucose, GOD is highly susceptible to oxygen in the blood, thereby resulting in an inaccurate measurement result. However, GDH is not interfered by oxygen in the blood, and thus are widely applied.
For GDH, it can be roughly classified into pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH), flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) and nicotinamide adenine dinucleotide-dependent glucose dehydrogenase (NAD-GDH) according to differences in coenzymes of GDH. As one of GDHs, PQQ-GDH uses pyrroloquinoline quinone (PQQ) as a coenzyme.
The EC number of PQQ-GDH was previously EC 1.1.99.17, and was later changed to EC 1.1.5.2. Currently, scientists have found two types of PQQ-GDH (EC 1.1.5.2) in bacteria: the first is a membrane-bound PQQ-GDH (PQQ-mGDH); and the second is a soluble PQQ-GDH (PQQ-sGDH). The biochemical properties of these two PQQ-GDHs are quite different. PQQ-sGDH is only found in the periplasmic space of Acinetobacter bacteria, such as A. calcoacelicus and A. baumannii, with A. calcoacelicus as an example for illustration hereafter.
A. calcoacelicus contains the aforementioned two different PQQ-GDHs, wherein PQQ-mGDH is active only in bacterial cells, and PQQ-sGDH only shows activity outside bacterial cells. Moreover, there is no significant homology between their sequences. PQQ-sGDH is composed of two identical subunits, each containing 3 calcium ions and having a molecular weight of 50 kD, wherein the N-terminus of each subunit is a signal peptide composed of 24 amino acid residues, and the signal peptide is excised after being secreted into the periplasmic space. PQQ-sGDH can catalyze the oxidation of various monosaccharides and disaccharides into corresponding ketones, the ketones are further hydrolyzed to aldonic acids, and this enzyme can provide the electrons generated during the oxidation reaction to phenazine methosulfate (PMS), 2,6-dichlorophenolindophenol (DCIP), Wurster's blue, and short-chain ubiquinone molecules such as ubiquinone Q1 and ubiquinone Q2, and certain artificial electron acceptors such as N-methylphenazonium methyl sulfate, and conductive polymers, etc. As compared with PQQ-mGDH, PQQ-sGDH has good water solubility and a wider range of electron-acceptor specificity, and thus it is well-suited for use in glucose determination with a test strip or a glucometer.
However, the wild-type PQQ-sGDH of A. calcoaceticus also has its own drawback, namely poor substrate specificity: it can not only oxidize glucose, but also oxidize monosaccharide and disaccharide molecules (such as maltose, galactose, lactose, mannose, xylose and ribose) through oxidation reactions. Such reactivity may cause some patients with diabetes to obtain wrong measurement values when measuring their own blood glucose levels. In particular, when diabetic patients are subjected to an intravenous injection of a formulation including maltose, galactose or xylose, or an icodextrin-based peritoneal dialysis, the blood glucose level measured through a glucometer with use of PQQ-sGDH as an oxidase may erroneously rise, and if these patients are treated according to the wrong blood glucose level, it may result in abnormal hypoglycemia, coma and even death.